ABSTRACT Alterations in basic DNA-DNA interactions can result in severe multi-spectrum developmental disorders, cell aneuploidy, genome instabilities and cancer. For instance, destabilization of DNA-DNA interactions in cis (at the base of a looped DNA molecule) are thought to abolish chromatin compaction and the registration of DNA regulatory elements (enhancers, promoters, insulators) that deploy developmental transcription programs. Alternatively, destabilization of DNA-DNA interactions in trans (between sister chromatids that arise by chromosome replication) simultaneously abolishes the identity of sisters required for high fidelity chromosome segregation and also access to template DNA required for error-free DNA repair. Cohesin complexes are critical for both cis and trans DNA-DNA associations. Thus, cohesin pathway mutations directly lead to deregulation of developmental programs that result in severe and multi-spectrum birth defect maladies such as Roberts Syndrome (RBS), Cornelia de Lange Syndrome (CdLS) and Warsaw Breakage Syndrome (WBS) and also correlate tightly with numerous forms of cancer, aneuploidy and genotoxic sensitivities. Complicating analyses of cohesin regulation is that the vast majority of cohesins are deposited onto DNA, independent of nucleotide sequence, such that cohesins can tether together any two DNA segments and in any part of the cell cycle. What we know is that the Scc2 and Scc4 heterocomplex is essential for all cohesin deposition onto DNA regardless of sequence, tether conformation or cell cycle stage. Unfortunately, less is known regarding the recruitment of Scc2,4 to DNA than even cohesin ? analyses similarly impeded by findings that any DNA sequence can support Scc2,4 recruitment and in any stage of the cell cycle. This revised R15 Renewal Proposal is predicated on two exciting findings emanating from my lab. First, we recently identified Chl1 DNA helicase as a key regulator of Scc2 deposition. In Specific Aim 1, we will characterize in detail the chromatin-based upstream regulation of Scc2 deposition that we predict will result in unified model through which Scc2 recruitment to DNA occurs during transcription, DNA replication and repair. During support through a previous R15 award, we also succeeded in isolating cohesin functions in cis tethering from that of trans tethering (and vice versa). Moreover, we discovered that each is regulated by spatially and temporally distinct mechanisms - one of which involves PCNA. In Specific Aim 2 of this revised R15 Renewal Proposal, we will dissect the molecular basis through which PCNA independently regulates trans cohesin tethers.